Thyroid hormones, such as L-thyroxine (T4) and 3,5,3′-triiodo-L-thyronine (T3), and their analogs such as GC-1, DITPA, Tetrac and Triac, regulate many different physiological processes in different tissues in vertebrates. It was previously known that many of the actions of thyroid hormones are mediated by the thyroid hormone receptor (“TR”). A novel cell surface receptor for thyroid hormone (L-thyroxine, T4, T3) has been described on integrin αvβ3. The receptor is at or near, but functionally and topographically distinct from, the Arg-Gly-Asp (RGD) recognition site on the integrin. The αvβ3 receptor is not a homologue of the nuclear thyroid hormone receptor (TR), but activation of the cell surface receptor results in a number of nucleus-mediated events, including the recently-reported pro-angiogenic action of the hormone and fibroblast migration in vitro in the human dermal fibroblast monolayer model of wound-healing.
Tetraiodothyroacetic acid (Tetrac) is a deaminated analog of T4 that has no agonist activity at the integrin, but inhibits binding of T4 and T3 to the integrin and the pro-angiogenic action of agonist thyroid hormone analogs at αvβ3. Inhibition of the angiogenic action of thyroid hormone has been shown in the chick chorioallantoic membrane (CAM) model, in the vessel sprouting model involving human dermal microvascular endothelial cells (HDMEC), and in vivo in the mouse matrigel angiogenesis model. In the absence of thyroid hormone, Tetrac blocks the angiogenic activity of basic fibroblast growth factor (bFGF, FGF2), vascular endothelial growth factor (VEGF) and other pro-angiogenic factors. Tetrac is effective in the CAM, HDMEC, and mouse angiogenesis models. This inhibitory action of Tetrac is thought to reflect its influence on the RGD recognition site that is relevant to pro-angiogenic actions.
Evidence that thyroid hormone can act primarily outside the cell nucleus has come from studies of mitochondrial responses to T3 or T2, from rapid onset effects of the hormone at the cell membrane and from actions on cytoplasmic proteins. The description of a plasma membrane receptor for thyroid hormone on integrin αvβ3 has provided some insight into effects of the hormone on membrane ion pumps, such as the Na+/H+ antiporter, and has led to the description of interfaces between the membrane thyroid hormone receptor and nuclear events that underlie important cellular or tissue processes, such as angiogenesis and proliferation of certain tumor cells.
Circulating levels of thyroid hormone are relatively stable; therefore, membrane-initiated actions of thyroid hormone on neovascularization or on cell proliferation or on membrane ion channels—as well, of course, as gene expression effects of the hormone mediated by TR mentioned above—may be assumed to contribute to “basal activity” or setpoints of these processes in intact organisms. The possible clinical utility of cellular events that are mediated by the membrane receptor for thyroid hormone may reside in inhibition of such effect(s) in the contexts of neovascularization or tumor cell growth. Indeed, it has been shown that blocking the membrane receptor for iodothyronines with tetraiodothyroacetic acid (Tetrac), a hormone-binding inhibitory analog that has no agonist activity at the receptor, can arrest growth of glioma cells and of human breast cancer cells in vitro. Tetrac is a useful probe to screen for participation of the integrin receptor in actions of thyroid hormone.
Thyroid hormone can also stimulate the proliferation in vitro of certain tumor cell lines. Murine glioma cell lines have been shown to proliferate in response to physiological concentrations of T4 by a mechanism initiated at the integrin receptor and that is MAPK-dependent. In what may be a clinical corollary, a prospective study of patients with far advanced glioblastoma multiforme (GBM) in whom mild hypothyroidism was induced by propylthiouracil showed an important survival benefit over euthyroid control patients. In 2004, it was reported that human breast cancer MCF-7 cells proliferated in response to T4 by a mechanism that was inhibited by Tetrac. (See Tang et al., Endocrinology 145:3265-72 (2004)). A recent retrospective clinical analysis by Cristofanilli et al. showed that hypothyroid women who developed breast cancer did so later in life than matched euthyroid controls and had less aggressive, smaller lesions at the time of diagnosis than controls. (See Cristofanilli et al., Cancer 103(6):1122-28 (2005); Baldazzi et al. Urol Oncol 2010 [Epub ahead of print]; Schmidinger et al. Cancer 2010 [Epub ahead of print]; and Riesenbeck et al. World J. Urol 2010 [Epub ahead of print]). Thus, the trophic action of thyroid hormone on in vitro models of both brain tumor and breast cancer appears to have clinical support.
The ability of tetraiodothyroacetic acid (Tetrac) to inhibit the action of T4 and T3 at the integrin is shown in FIG. 1. Tetrac blocks the binding of iodothyronines to the integrin receptor. Also shown is crosstalk between the integrin and vascular growth fator receptors clustered with the receptor as well as the epidermal growth factor receptor (EGFR). Here, the presence of thyroid hormone at the cell surface alters the function of EGFR to allow the latter to distinguish EGF from TGF-α, another growth factor that binds to EGFR. Moreover, the receptor is able to distinguish among the different thyroid hormone analogs and to selectively activate MAPK or PI3K, depending on the particular analog that this bound to the receptor.
There is a need in the art for thyroid hormone analogs that can bind to the cell surface receptor while not being able to enter the cell. Such reformulated hormone analogs would not express intracellular actions of the hormone and, thus, if absorbed into the circulation, would not have systemic thyroid hormone analog actions.